The effects of reactive oxygen species (ROS) and oxidative stress on protein organization, cellular homeostasis, and apoptosis are recognized as key factors in aging and senescence, and ROS damage is thought to play a role in a number of neurodegenerative diseases including Alzheimer's, Parkinson's Disease, and stroke (ischemia/reperfusion). The turtle, an accepted model of aging with negligible senescence, has provided new insights into the defense against oxidative stress as certain species survive prolonged bouts of anoxia and repeated reoxygenation without apparent cellular damage. This ability of the freshwater turtle (Trachemys scripta) is due both to high constitutive levels of antioxidants and an inherent ability to suppress excess ROS formation (Milton et al., 2007), and we hypothesize that the ability of the turtle to prevent oxidative damage is directly linked to its aging without senescence. This species is also unique in the animal kingdom in that it significantly upregulates the enzyme peptide Methionine sulfoxide reductase (MsrA) in the brain during anoxia. MsrA and MsrB are "repair enzymes" for oxidized methionine residues on proteins and free amino acids;oxidized methionine compromises the activity of a number of enzymes and Msr may thus restore biological activity to damaged proteins. The reversible oxidation/reduction of readily available Met in proteins may also be one of the prime mechanisms by which cells catalytically scavenge ROS before they damage cellular constituents (Levine et al., 1996), and increased cell death and ROS damage occur when Msr levels are reduced in several aging models. This is the first report in any system -eukaryotic or prokaryotic- of the induction and subsequent down-regulation of MsrA transcription and protein levels regulated by oxygen supply, which makes T. scripta radically different from other animal models, and provides a unique opportunity to investigate the function and regulation of this peptide which is likely to play a critical role in aging. Heat shock protein 72 (Hsp72) is also strongly upregulated by anoxia, and its role as a molecular chaperone to reduce accumulation of AGE products as well as an anti-apoptotic protein may also play an important role in aging without senescence. HSP's are involved in ischemic preconditioning, neuroprotection in ischemia and stroke, and act as end-effectors of anti-apoptotic mechanisms. Our specific aim is to determine how MsrA and Hsp72 protect cells against oxidative damage;we will utilize in vivo studies as well as siRNA technology in vitro to study the role of MsrA and Hsp72 in neuronal resistance to oxidative stress, cellular damage, and mitochondrial dysfunction, and thus their role in aging without senescence. PUBLIC HEALTH RELEVANCE: Reactive oxygen species (ROS) and oxidative stress affect protein organization, cellular homeostasis, and apoptosis, and are recognized as key factors in aging and senescence, as well as neurodegenerative diseases including Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS) and stroke (ischemia/reperfusion). Methionine sulfoxide reductases are a ubiquitous class of enzymes that repair oxidative damage to methionine residues in proteins, and which are uniquely upregulated by anoxia in the brain of the anoxia-tolerant turtle;heat shock proteins are also strongly upregulated and defend against ROS release and protein damage This project utilizes the turtle as a model to investigate the role of MsrA and Hsp72 in neuronal resistance to oxidative stress, cellular damage, and mitochondrial dysfunction, and thus its role in aging without senescence.